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WO1997012988A1 - Procede et compositions faisant intervenir un heterodimere bispirale pour la detection et la purification de proteines exprimees - Google Patents

Procede et compositions faisant intervenir un heterodimere bispirale pour la detection et la purification de proteines exprimees Download PDF

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Publication number
WO1997012988A1
WO1997012988A1 PCT/US1996/016032 US9616032W WO9712988A1 WO 1997012988 A1 WO1997012988 A1 WO 1997012988A1 US 9616032 W US9616032 W US 9616032W WO 9712988 A1 WO9712988 A1 WO 9712988A1
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WIPO (PCT)
Prior art keywords
heterodimer
subunit
peptide
seq
coil
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PCT/US1996/016032
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English (en)
Inventor
Michael E. Houston, Jr.
Lei Yu
Daisy Bautista
Robert S. Hodges
Brian Tripet
Paul J. Cachia
Randall T. Irvin
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Pence
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Application filed by Pence filed Critical Pence
Priority to JP9514507A priority Critical patent/JPH11512620A/ja
Priority to CA002234073A priority patent/CA2234073C/fr
Priority to AU72584/96A priority patent/AU695679B2/en
Priority to EP96934080A priority patent/EP0854931A4/fr
Publication of WO1997012988A1 publication Critical patent/WO1997012988A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • COILED-COIL HETERODIMER METHODS AND COMPOSITIONS FOR THE DETECTION AND PURIFICATION OF EXPRESSED PROTEINS
  • This invention relates to the use of a pair of peptides capable of forming ⁇ -hehcal coiled-coil heterodimers for the purification and detection of fusion proteins containing one of the peptides of the pair
  • the invention includes an expression vector composed of a replication segment which permits replication of the vector in a selected host cell, and an expression cassette
  • the cassette contains, in a 5'-3' direction, a promoter functional in the host cell and a coding segment
  • the coding segment contains a heterologous DNA coding sue and a DNA region encoding a first heterodimer-subunit peptide capable of forming an ⁇ - helical coiled-coil heterodimer with a complementary second heterodimer-subunit peptide
  • the coding segment may contain, disposed between the coding site and the DNA region, a cleavage sequence, in frame with the DNA region and encoding an ammo acid sequence that provides a target for chemical or enzymatic cleavage
  • the coding segment may be oriented with either its coding site or DNA region adjacent the promoter
  • one of the (first and second heterodimer-subunit peptides) contains at least two heptad am
  • the cloning site preferably includes a multiple cloning site (MCS) at which a heterologous DNA coding region can be inserted
  • MCS multiple cloning site
  • a heterologous DNA coding region typically encodes a selected polypeptide of interest
  • the DNA encoding the selected polypeptide is typically inserted at the cloning site such that it is expressed in frame with coding sequences in the DNA region encoding the first heterodimer-subunit peptide are the sequences represented by SEQ ID NO 2 and SEQ ID NO 4
  • the invention includes a coding segment, for use in an expression vector suitable for expressing heterologous proteins in a host cell (such as the vector described above)
  • the segment includes a heterologous DNA coding sue and a DNA region encoding a heterodimer-subunit peptide capable of forming an ⁇ -helical coiled-coil heterodimer with a complementary heterodimer-subunit peptide, as described above Further, the segment
  • a reagent for detecting the presence of the expressed fusion protein is composed of a second heterodimer-subunit peptide (second coil peptide) capable of forming an ⁇ -helical coiled-coil heterodimer with the heterodimer-subunit peptide (first coil peptide) in the fusion protein
  • the reagent is useful for detecting expression of a fusion protein containing a heterodimer-subunit peptide, such as described above, by forming a heterodimer or heterodimer complex between the heterodimer-subunit peptide contained in the fusion protein and the detection reagent, and detecting the presence of heterodimer formation
  • a kit for detecting the expression of a selected polypeptide includes an expression vector containing the above-described coding segment, for expressing a fusion protein containing the heterodimer-subunit peptide, and the above detection reagent
  • the invention includes an affinity matrix composition for the purification of a selected polypeptide
  • the composition includes a solid support, and attached to said support, a heterodimer-subunit peptide (coil peptide, e g , SEQ ID NO 4 or SEQ ID NO 27) of the type described above
  • the composition is useful in a method for purifying a selected expressed polypeptide, as described below
  • the invention also includes a method of purifying a selected expressed polypeptide
  • the method includes the steps of (I) expressing, in a suitable host-cell expression system (e g , an E coli or Yeast expression system), a fusion protein containing the selected polypeptide in tandem with a first heterodimer subunit (e g , SEQ ID NO 2 or SEQ ID NO 26), (n) obtaining, from the host cell expression system, a suspension containing said fusion protein, and (in) passing this suspension over an affinity matrix composition as described above The affinity matrix is then washed while the fusion protein remains bound to the affinity mat ⁇ x via the ⁇ -hehcal coiled-coil heterodimer
  • the washing includes, in either order, (a) a salt wash step comprising washing the affinity matrix with a solution having a pH of between about 5 5 and about 8 0 and containing between about 0 1 M and about 1 M salt (e g , sodium chloride, potassium chloride, sodium acetate, ammonium
  • a kit for obtaining an expressed protein in purified form includes the above expression system, for expressing a fusion protein having a heterodimer-subunit peptide of the type described above, and an affinity matrix composition of the type described above, for affinity binding of the expressed fusion protein to the solid support in the composition
  • FIGS. IA, I B, IC, I D I E and IF show a scheme for affinity purification according to the method of the invention
  • Figure 2 shows helical wheel representations of peptides 0993 (SEQ ID NO 26) and 0994 (SEQ ID NO 27) in an ⁇ -helical heterodimer configuration
  • Figures 3A, 3B, 3C, 3D and 3E show a schematic representations of adjacent heptads of two heterodimer-subunit peptides in a parallel configuration comparing the stabilizing/destabilizing effects of charged residues at the e and g positions in homodimers vs heterodimers
  • Figure 3A shows a homodimer stabilized by oppositely-charged residues at the e and g positions of a heptad
  • Figure 3B shows a heterodimer destabilized by oppositely-charged residues at the e and g positions of a heptad
  • Figure 3C shows a homodimer destabilized by positively-charged residues at the e and g positions of a heptad
  • Figure 3D shows a heterodimer stabilized by like-charged residues at the e and g positions of a heptad
  • Figure 3E shows a homodimer destabilized by negatively-charged
  • Figures 4A, 4B and 4C show a schematic of some possible distributions of heptads, bearing either positive or negative charges at their e and g positions, within peptides designed to form coiled-coil heterodimers
  • Figure 4A shows a schematic of a heterodimer comprised of heterodimer-subunit peptides having alternating positively- and negatively- charged successive heptads
  • Figure 4B shows a schematic of a heterodimer comprised of heterodimer-subunit peptides, one of which has predominantly positively-charged heptads, and the other of which has predominantly negatively-charged heptads
  • Figure 4C shows a schematic of a heterodimer comprised of heterodimer-subunit peptides.
  • Figures 5A, 5B, 5C, 5D, 5E, 5F and 5G show a schematic of the synthesis of a
  • FIG. 5H shows a map of plasmid pRLD-E
  • Figure 6A shows an expression vector expression cassette useful for expressing a fusion protein having a heterodimer-subunit peptide at the N-terminus of a selected protein encoded by a DNA fragment inserted at the multiple cloning site (MCS)
  • Figure 6B shows an expression vector expression cassette useful for expressing a fusion protein having a heterodimer-subunit peptide at the C-terminus of a selected protein encoded by a DNA fragment inserted at the multiple cloning site (MCS)
  • Figure 7A shows a reversed-phase (RP)-HPLC chromatogram of peptide 0993 (SEQ ID NO:26).
  • Figure 7B shows an RP-HPLC chromatogram of the injection flow through fraction of a selective dimerization affinity column of the invention.
  • Figure 8 shows an RP-HPLC chromatogram of the 60% acetonitrile wash fraction of the affinity column described in Figure 7B.
  • Figure 9 shows an RP-HPLC chromatogram of a subsequent GndHCl elution fraction of the affinity column described in Figure 8.
  • Figure 10 shows a chromatogram of a mixture of three test charged peptides (SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30) used to test specificity of the affinity matrix of the invention.
  • Figure 1 1 shows a chromatogram of the 0.2 M KCl wash fraction of an affinity column onto which test peptides depicted in Figure 10 were loaded.
  • Figure 12 shows a chromatogram of the 0.5 M KCl wash fraction of an affinity column to which test peptides were loaded.
  • Figure 13 shows a chromatogram of the 1.0 M KCl wash fraction of the affinity column described in Figure 12.
  • Figure 14 shows a chromatogram of a subsequent 1 .0 M KCl wash of the column described in Figure 13.
  • Figure 15 shows a subsequent 5.0 M GndHCl/50mM K P0 4 of the column described in Figure 14.
  • Figure 16A shows an SDS-PAGE gel of PAK( 128- 144)/E-coil fusion protein stained with comassie blue.
  • Figure 16B shows western and ligand blots of PAK( 128- 144)/E-coil fusion protein probed with either coil K reporter or monoclonal antibody PK99H .
  • Figure 17 shows a series of RP-HPLC chromatograms of load samples and fractions eluted from an affinity column of the invention, where the load fraction was a crude periplasmic extract containing recombinantly expressed E-coil peptide.
  • Figure 18 shows a series of RP-HPLC chromatograms of purification of recombinantly expressed Pak-pili-E-coil peptide from crude on an affinity column made in accordance with the present invention.
  • Figure 19 shows an ELISA assay employing a reporter molecule formed according to the invention. Detailed Description of the Invention
  • ' peptide designates a chain of amino acid based polyamides
  • the chain can vary in length anywhere from 2 amino acids to about 100 ammo acids
  • polypeptide designates a chain of ammo acid based polyamides containing at least 2 ammo acids
  • a promoter functional in a host means a DNA sequence in an expression vector effect to promote transcription of an adjacent 3' downstream coding sequence in the vector in a selected host
  • a "coding segment" in an expression vector refers to a segment of DNA which encodes a polypeptide
  • heterologous DNA coding site refers to (I) a rest ⁇ ction sue or sues, e g , multiple cloning site, in an expression vector, into which a DNA that encodes a selected heterologous protein can be inserted, or (n) the encoding DNA itself
  • heterodimer-subunit peptide refers to one of two complementary peptide subunits which are capable of forming a coiled-coil heterodimer under selected conditions
  • heterodimer polypeptide or “heterodimer polypeptide complex” refers to two associated non-identical polypeptide chains Unless otherwise indicated, the sequence for peptides and polypeptides is given in the order from the am o terminus to the carboxyl terminus All amino acid residues identified herein are in the natural or L-configuration unless otherwise specified In keeping with standard peptide nomenclature, abbreviations for amino acid residues are standard 3- letter and/or 1 letter codes commonly used in the art
  • the term "derivatized” in the context of a "derivatized” polypeptide subunit, is understood to refer to a polypeptide subunit having one or more functional or reporter moieties covalently attached to one or more amino acid residues forming the subunit, where the moiety
  • benign medium describes a physiologically-compatible aqueous solution typically having a pH of between about 6 and about 8 and a salt concentration of between about 50 mM and about 500 mM. Preferably, the salt concentration is between about 100 mM and about 200 mM.
  • An exemplary benign medium, designated as buffer A. has the following composition: 50 mM potassium phosphate, 100 mM KCl, pH 7. Equally effective benign media may be made by substituting, for example, sodium phosphate for potassium phosphate and/or NaCl for KCl.
  • Complementary heterodimer-subunit peptides are two non-identical peptide chains, typically about 21 to about 70 residues in length, having an amino acid sequence compatible with their formation into two-stranded ⁇ -helical heterodimeric coiled-coils. They are designated herein generally as HSP1 (heterodimer-subunit peptide 1 ), and HSP2 (heterodimer-subunit peptide 2). Because the peptides can form ⁇ -helical coils, they are also referred to herein as "coil peptides".
  • HSP1 and HSP2 are mixed together under conditions favoring the formation of ⁇ -helical coiled-coil heterodimers, they interact to form a two-stranded ⁇ - helical coiled-coil heterodimeric complex, designated as HSP1 - HSP2.
  • Peptides in an ⁇ -helical coiled-coil conformation interact with one another in a characteristic manner that is determined by the primary sequence of each peptide:
  • the tertiary structure of an ⁇ -helix is such that 7 amino acid residues in the p ⁇ mary sequence correspond to approximately 2 turns of the ⁇ -helix. Accordingly, a primary amino acid sequence giving rise to an ⁇ -helical conformation may be broken down into units of 7 residues each, termed heptads.
  • the heterodimer-subunit peptides are comprised of a series of heptads in tandem. When the sequence of a heptad is repeated in a particular heterodimer-subunit peptide, the heptad may be referred to as a "heptad repeat", or simply "repeat" .
  • HSP1 and HSP2 may also contain residues that can be reacted (either intra- or inter- helically) to stabilize the ⁇ -helical or coiled-coil nature of the polypeptides.
  • Complementary heterodimer-subunit peptides are typically of similar size, each generally ranging from about 21 to about 70 residues (3 to 10 heptads) in length
  • the peptides may be synthesized by a variety of methods known to those skilled in the art, including chemical and recombinant methods.
  • an ABI Model 430A peptide synthesizer may be used with conventional t-Boc chemistry as described previously by Hodges, et al.. ( 1988), and in Example 1.
  • the peptides may be expressed in an appropriate host-cell system using an expression vector encoding the peptides, as detailed, e.g. , in Examples 5 and 6.
  • the peptides may be purified by any of a number of methods known to those skilled in the art, for example using reversed-phase high performance liquid chromatography (RPC) and a "SYNCHROPAK” RP-P column, as detailed in Example 1.
  • RPC reversed-phase high performance liquid chromatography
  • SYNCHROPAK SYNCHROPAK
  • composition and purity of the peptides can be verified by several methods, including amino acid composition mass analysis on a Beckman model 6300 amino acid analyzer and molecular weight analysis using time of flight mass spectroscopy on a "BIOION-20" Nordic, as detailed in Example 1.
  • HSP1 and HSP2 The dimerization of HSP1 and HSP2 occurs due to the presence of a repeated heptad motif of conserved amino acid residues in each peptide's primary amino acid sequence.
  • the individual positions in each heptad are designated by the letters a through g for HSP1. and a' through g for HSP2, as shown in Figure 2.
  • the positions (e.g.. a', g') of HSP2 are sometimes referred to without the (') symbol in general discussions of heptad positions in heterodimer-subunit peptides, or coil peptides, below.
  • HSP1 and HSP2 polypeptides Repeating heptad motifs having appropriate amino acid sequences direct the HSP1 and HSP2 polypeptides to assemble into a heterodimeric ⁇ -helical coiled-coil structure under permissible conditions, presented in part D, below.
  • the individual ⁇ -helical peptides contact one another along their respective hydrophobic faces, defined as the a and d positions of each heptad.
  • HSP1 and HSP2 may assemble into a heterodimer coiled-coil helix (coiled-coil heterodimer) in either parallel or antiparallel configurations.
  • the two heterodimer-subunit peptide helixes are aligned such that they have the same orientation (amino-terminal to carboxyl-terminal).
  • the helixes are arranged such that the amino-terminal end of one helix is aligned with the carboxyl-terminal end of the other helix, and vice versa
  • Figure 2 shows an end-on schematic of the first two turns (one heptad) of two exemplary heterodimer-subunit peptides.
  • 0993 E, SEQ ID NO 26
  • 0994 K SEQ ID NO 27
  • Heterodimer-subunit peptides designed in accord with the guidance presented herein typically show a slight preference for assembling in a parallel orientation vs an antiparallel orientation Generally, however, the orientation (parallel vs antiparallel) in which the two heterodimer-subunit peptides form an ⁇ -helical coiled coil is not particularly relevant to their ability to hold together moieties attached to the heterodimer-subunit peptides
  • hydrophobic interactions between the helixes are due to hydrophobic residues at the a and d positions of the heterodimer-subunit peptides Residues at these positions, effective to maintain the helixes in contact, include leucine, isoleucine. valine, phenylalanme. methionine, tryptophan, tyrosine, alanine and derivatives of any of the above Other residues, including alanine, cysteine, serine. threonine. asparagine and glutamine may also occupy a or d positions in some heptads, so long as others are occupied by hydrophobic residues
  • residues at the a and d positions are selected such that the hydrophobic interactions are too weak (for example, Ala at both positions), the helixes may not form coiled-coil dimers at all
  • residue pairs at the a and d positions are selected to promote the formation 95% heterodimers at pH 7, more preferably > 99% heterodimers.
  • the degree of heterodimer vs. homodimer formation may be measured as described. for instance, in Example 3.
  • results of experiments performed in support of the present invention indicate that if all the a and d positions in all heptads of both coil peptides are occupied by Ile. Leu. or any combination thereof, the hydrophobic interactions will be strong enough to stabilize homodimers as well as heterodimers. even if the e and g positions contained charged residues designed to stabilize heterodimers as detailed herein. Further, such homodimers will form among coil peptides containing as few as three heptads. According to an important aspect of the invention, the formation of such homodimers should be minimized or, for practical purposes, eliminated.
  • the coil peptides in the different component solutions i.e. , the coil peptides in a suspension containing fusions of a selected protein and a coil peptide, or the coil peptides in a suspension to be used for derivatizing the solid matrix
  • the coil peptides in the different component solutions would self-assemble before the component solutions could be used for their intended purpose.
  • Several strategies may be employed to modulate or set the strength of hydrophobic interactions among residues at the a and d positions to a point where the interactions will be strong enough to maintain heterodimers during the protein purification procedure, yet not so strong that they will stabilize homodimers.
  • An preferred strategy is to use coil peptides where all heptads have Leu at position d, and Val at position a, or coil peptides where all heptads have Ile at position d. and Val at position a.
  • Another strategy is to use to use coil peptides where all heptads have Leu at position a.
  • the conformation of polypeptides, such as HSP1 and HSP2 in solution can be determined from CD spectra of the solution. These data provide information as to the conformation of the individual peptides themselves (random coil vs. ⁇ -helical). as well information as to the relative amounts of heterodimer vs. homodimer complexes of, for example, HSP1 and HSP2.
  • Example 2 details one method of measuring CD spectra.
  • Example 3 details how a CD spectra measurements can be used to assess the conformation of peptides in solution.
  • ionic interactions between the two helixes arise from negatively-charged (Glu) residues at the e and g positions on HSP1 (0993; SEQ ID NO:26), and positively-charged (Lys) residues at the e and g positions on HSP2 (0994; SEQ ID NO:27).
  • Negatively-charged residues can be aspartic acid, glutamic acid or derivatives thereof.
  • Positively-charged residues can be lysine, arginine. histidine. or derivatives thereof.
  • Heterodimer-subunit peptides comprised of repeating heptads and designed according to the guidance presented in parts A through C. above, will readily form coiled- coil heterodimers in a benign medium, defined above in part 1.
  • the degree of ⁇ -helical coiled-coil heterodimer formation can be determined from CD spectra, as described, for instance, in Example 3.
  • Coiled-coil heterodimers may form under conditions outside the pH and salt range given for a benign medium, but some of the molecular interactions and relative stability of heterodimers vs. homodimers may differ from characteristics detailed above. For example, ionic interactions between the e and g positions that tend to stabilize heterodimers may break down at low or high pH values due to the protonation of, for example, Glu side chains at acidic pH, or the deprotonation of, for example, Lys side chains at basic pH.
  • This part describes some examples of how heptads with sequences which are in compliance with the guidelines presented in parts A through C, above, can be arranged within the heterodimer-subunit peptides
  • Heterodimer-subunit peptides of the present invention may each contain from three to a plurality of heptads
  • the sequences of each of those heptads may all be the same, or they may differ
  • the sequences of the internal repeats may differ from one another depending on, for example, whether or not the repeats incorporate amino acid coupling residues, such as cysteine
  • Such a coupling residue may be used, for example, to anchor a heterodimer-subunit peptides to a resin support mat ⁇ x or to another polypeptide
  • the primary sequence of heptads within a heterodimer-subunit peptide can vary, so long as the residues within each heptad interact favorably with residues in the complementary heptad of the second heterod
  • adjacent heptads may vary in sequence such that, for example, the net charge on the heterodimer-subunit peptides can be altered without affecting the ability of the polypeptides to form ⁇ -helical heterodimer coiled-coils
  • Figure 4 The figure shows three examples of CP dimer pairs Each heterodimer-subunit peptide has 5 heptads
  • the + or - symbols in each heptad each represent two charges (one at the e position and one at the g position)
  • adjacent complementary heptads have opposite charges
  • HSPl and HSP2 forming the dimer in Figure 4A have net charges of +2 and -2, respectively due to an excess of one positively-charged heptad.
  • HSPl and HSP2 in Figure 4B have net charges of +6 and -6, respectively
  • HSPl and HSP2 in Figure 4C have net charges of + 10 and -10 respectively
  • Other variations on this theme are. of course, possible without departing from the spirit of the invention
  • Coupling residues such as amino-acid coupling residues, may be inco ⁇ orated into one or both coil peptides to facilitate subsequent uses of the peptides
  • the coupling residues may be inco ⁇ orated at in the central region of the peptide and/or at one or both ends In cases where the coupling residues are in the central region, the residues are typically inco ⁇ orated at positions b, c and/or f, preferably at position f, of one or more heptads These positions lie along the outward face of a coiled-coil heterodimer Coupling residues at one or both ends are typically coupled to the terminal residue
  • Preferred couphng groups are the thiol groups of cysteine residues, which are easily modified by standard methods Example 4 details how the cysteine thiol groups present in coil peptides can be used to attach other peptides at those positions
  • Other useful coupling groups include the thioester of methionine, the imidazolyl group of histidine, the guamdiny l group of arginine. the phenolic group of tyrosine and the mdolyl group of tryptophan These coupling groups can be derivatized in a manner similar to that detailed in Example 4, using reaction conditions known to those skilled in the art
  • E-coil (SEQ ID NO 2) and K-coil (SEQ ID NO 4) heterodimer-subunit peptides are exemplary HSPl and HSP2 heterodimer-subunit peptides Both peptides contain Val residues at their a positions, and Leu residues at their d positions, ensuring hydrophobic interactions effective to stabilize coiled-coil heterodimers, but not strong enough to overcome the electrostatic repulsion between homodimers
  • each of the corresponding d/a' and a/d' pairs in such complementary heptads consists of residues where one of the residues is a valine and the other is selected from the group consisting of leucine and isoleucine
  • the d/a' pair consists of Leu (at the d position) and Val (at the a' position)
  • the a/d' pair consists of Val (at the a position) and Leu (at the d' position)
  • the invention includes an expression vector useful for expressing a fusion protein of a selected polypeptide expressed in tandem with a coil peptide (I e, a heterodimer-subunit peptide)
  • the coil peptide portion of the expressed fusion may then be used to purify the protein using an affinity matrix (as described below), or to detect the protein, either in situ (e g . fluorescence detection in a cell) or in a protein detection system, such as enzyme- linked immunosorbent assay (ELISA). and/or a dot. slot or Western blot
  • the fusion protein is detected using a detection reagent which includes, e g , a reporter-labeled coil peptide complementary to the coil peptide in the fusion
  • Such plasmids or vectors may then be transformed into suitable host cells, such as bacteria or yeast, and the cells induced to produce recombinant polypeptides Depending on the expression system, the medium in which the cells were induced, or the cells themselves, are used to make a suspension containing the recombinant polypeptide, which may then be purified using the methods of the invention
  • Example 5 details the recombinant construction of the E-coil gene using two sets of complementary overlapping oligonucleotides
  • the oligonucleotides in each set were annealed, digested with the appropriate rest ⁇ ction enzymes to create sticky ends at one end of each double-stranded (ds) fragment (Figs. 5C and 5F), and the two ds fragments were ligated with T4 DNA Ligase to generate a single ds fragment (Fig. 5G) of 177 base pairs.
  • the 177 bp fragment was purified and digested with EcoRI and BamH] to produce a 166bp fragment suitable for cloning into a plasmid vector.
  • the construction of an exemplary plasmid expression vector, pRLD. is described in
  • Example 6 pRLD was constructed by changing the polylinker site of expression vector pASK40 (Skerra, et al.. 1991 ) to correspond to the polylinker of pHIL-Sl and PIC9 (Invitrogen, San Diego, CA). This modification was designed to shift the reading frame and make pASK40's cloning site more consistent with that of Pichia pastoris vectors. A sequence encoding the E-coil gene was then cloned into pRLD to produce the E. coli E-coil vector pRLD-E ( Figure 5H).
  • the expression vector may contain a replication segment which permits autonomous replication of the vector in a selected host cell, and an expression cassette.
  • the cassette contains, in a 5'-3' direction, a promoter functional in the host cell and a coding segment, which is described in detail, below.
  • the vector may also contain additional elements, such as sequences encoding a selectable marker that assure maintenance of the vector in the cell, an appropriately positioned ribosome-binding site downstream of the promoter, and transcription termination (TT) sequences at the 3' end of the cassette.
  • elements typically contained in E. coli expression vectors depend on the particular expression system with which the vectors are used.
  • elements typically present in E. coli expression vectors include (i) sequences encoding a selectable marker that assure maintenance of the vector in the cell (e.g. , an ampicillin resistance gene), (ii) a controllable transcriptional promoter which, upon induction, can produce large amounts of mRNA from a selected gene cloned into the vector, (iii) translational control sequences, such as an appropriately positioned ribosome-binding site and initiator ATG, and (iv) a polylinker or multiple cloning site (MCS) to simplify the insertion of the selected gene in the correct orientation within the vector.
  • a selectable marker that assure maintenance of the vector in the cell
  • a controllable transcriptional promoter which, upon induction, can produce large amounts of mRNA from a selected gene cloned into the vector
  • translational control sequences such as an appropriately positioned ribo
  • inducible promoters suitable for use with E. coli expression vectors include lac, trp, and tac, which, upon induction, can produce large amounts of mRNA from genes operatively linked to the promoters.
  • Other promoters suitable for use with E. coli expression vectors include the highly-efficient phage T7 gene 10 promoter, which uses T7 RNA polymerase, and the powerful bacteriophage pL promoter (Ausubel, et ai , 1988).
  • Recombinant polypeptides or fusion proteins are typically expressed by transforming a suitable E. coli expression strain (e.g. , JM83) with expression plasmids encoding recombinant polypeptides or fusion proteins
  • suitable transformation methods include heat-shock treatment (Yanisch-Perron. et al , 1985) and electroporation
  • the transformed cells are then typically plated on selective agar plates ( ⁇ ? g . LB plates supplemented with carbenicillin ( 100 ⁇ g/ml)), and cells containing the approp ⁇ ate recombinant DNA are verified by restriction digest analysis and subsequent DNA sequencing of minipreped plasmid DNA
  • Selected E coli clonal cells harboring the appropriate DNA are typically grown (e g , at 25 °C with shaking in LB medium containing 100 ⁇ g/ ⁇ l of carbenicillin) to a suitable density (e g , an A 550 of 0 5)
  • a suitable density e g , an A 550 of 0 5
  • the cells are induced by addition of the appropriate inducing agent
  • a suspension containing the expressed protein may be obtained using methods known in the art, e g , a modified osmotic shock treatment (Ausubel, et al , 1988) Although proteins expressed in large amounts in E coli sometimes precipitate into insoluble aggregates (inclusion bodies), such proteins may be recovered using, for example, solubilization in denaturing agents known to those skilled in the art (Ausubel.
  • yeast such as Pichia pastoris
  • yeast expression vectors may be used to transform yeast spheroplasts, and the transformed cells used to produce recombinant polypeptide
  • Exemplary inducible promoters suitable for use with yeast expression vectors are the AOX promoter, described above, and the GALL GAL4, GAL7 and the GAL10 promoters
  • Example 7 describes the use of a yeast expression vector in the practice of the invention
  • a DNA fragment containing the E-coil gene was PCR-amplified and cloned into plasmid pIC9 (Invitrogen, San Diego, CA), generating Pichia E-coil vector p9CE Verified yeast plasmids were transformed into Pichia pastoris, and the yeast cells were selected, induced and processed as described in Example 7 to generate culture superna
  • Expression cassettes or coding segments of the present invention may also be used to express fusion proteins usmg the baculovirus system, where genes for proteins to be expressed are inserted into an insect virus in lieu of a highly expressed dispensable gene
  • the foreign protein is produced by growing the recombinant virus in cultured insect cells (Ausubel, et al , 1988).
  • Such vectors typically contain recombination sequences to allow the vector to integrate into the host cell's genome via homologous recombination
  • the specific promoters used in these vectors generally depend on the identity of the excised dispensable gene. Exemplary promoters include the polyhedrin promoter (normally used to drive expression of the AcMNPV polyhedrin gene) and the plO promoter (Ausubel. et ai , 1988).
  • Mammalian cells may also be used as host cells for vectors employing expression cassettes or coding segments of the present invention.
  • Two exemplary mammalian systems employ the COS and CHO cell lines, respectively, ln the COS system, vectors containing the gene to be expressed are transiently transfected into COS cells, which constitutively produce SV40 large T antigen.
  • Vectors used with COS cells typically contain an SV40 replication origin, such that when the vectors are transfected into COS cells, they replicate and thereby amplify the amount of protein expressed.
  • CHO cells may be stably transfected with vectors employing expression cassettes or coding segments of the present invention and carrying either the dihydrofolate reductase or the glutamine synthetase gene (whose products confer drug resistance).
  • Cell lines that carry increased numbers of the constructs are obtained by selecting those that grow in increasing drug concentrations (e.g. , methotrexate). Once selected, these lines are permanent reagents, which can be stored frozen and used to produce the protein whenever desired.
  • the coding segment contains a heterologous DNA coding site, a DNA region encoding a first heterodimer-subunit peptide capable of forming an ⁇ -helical coiled-coil heterodimer with a complementary second heterodimer-subunit peptide. and disposed between the coding site and the DNA region, a cleavage sequence.
  • the cleavage sequence is in frame with the DNA region, and encodes an amino acid sequence that provides a target for chemical or enzymatic cleavage.
  • the cleavage sequence is preferably included in coding segments of the present invention to facilitate the removal of the coil peptide from its fusion partner (the selected polypeptide of interest).
  • the invention contemplates a coding segment containing only a heterologous DNA coding site and a DNA region encoding a first heterodimer-subunit peptide capable of forming an ⁇ -helical coiled-coil heterodimer with a complementary second heterodimer-subunit peptide.
  • the coding site is a multiple cloning site (MCS), also referred to as a polylinker or polylinker site.
  • MCS multiple cloning site
  • the MCS typically contains a number of restriction enzyme sites useful for cloning selected polynucleotides to be expressed into the coding segment or vector containing the coding segment Examples of suitable poly nkers can be found in virtually all commercially-available cloning vectors (e g . vectors from Stratagene. La Jolla, CA or Invitrogen. San Diego, CA)
  • the coding site contains a gene encoding a protein of interest
  • a gene encoding PAK pi antigen is generated by annealing synthetically-produced complementary oligonucleotides and cloning the ds fragment into appropriate restriction sues in the pRLDE vector
  • a cDNA encoding GFP is cloned into pRLDE
  • Exemplary proteins which may be purified using the methods of the present invention include epoetm ⁇ , G-CSF, somatotropic, insulin, tPA, urokinase and prourokinase, va ⁇ ous interferons, EPO, and various specialty enzymes
  • the coding segment may contain the coding site oriented either 5' or 3' of DNA region
  • Figs 6A and 6B show schematics of expression cassettes of the present invention
  • the cassettes contain a promoter functional in the selected host cell system, and, disposed at the 3' ends of the promoter regions, coding segments as described above
  • the heterologous DNA coding sites are represented as "cDNA”
  • the DNA regions encoding a first heterodimer-subunit peptide are represented as "Coil DNA”
  • Figure 6A shows the coding sue oriented at the 3' end of the DNA region
  • Figure 6B shows the coding sue oriented at the 5' end of the DNA region
  • proteases Cleavage sites that constitute targets for rare-cutting proteases are known in the art (Ausubel. et al . 1988) Such proteases mclude factor Xa (Nagai and Thogersen, 1984, 1987), thrombin (Smith and Johnson, 1988, Gearing, et al , 1989), enterokinase (Dykes, et al , 1988, LaVallie.
  • Factor Xa and enterokinase are particularly useful because they cleave on the carboxy-terminal side of their respective recognition sequences, allowing the release of fusion partners containing their authentic ammo-termini
  • the recognition sites for enterokinase, factor Xa and thrombin are provided herein as SEQ ID NO 21 , SEQ ID NO 23 and SEQ ID NO.25, respectively
  • fusions of a peptide such as a coil peptide
  • a selected polypeptide of interest sometimes alter a desired activity or characteristic of the polypeptide of interest
  • the probability of obtaining a fusion that does not significantly alter a desired property or characteristic of the selected polypeptide of interest is increased
  • Exemplary coding sequences for the DNA region encodmg the first heterodimer- subunit peptide are the sequences represented by SEQ ID NO 2, SEQ ID NO 4.
  • Coding segments of the present invention are preferably provided in three configurations for each of the two arrangements of elements described above
  • the three forms each differ from one another by having different-length nucleotide spacers between the coding site and the cleavage site
  • the spacers are included to provide coding segments having the coding sue in all three reading frames relative to the reading frame of the DNA region and the cleavage site
  • an appropriate coding segment can be selected by those of skill in the art to express any DNA fragment having ends compatible with rest ⁇ ction sues present at the MCS in frame with the DNA region and the cleavage sue
  • V Affinity Matrix Composition Employing Heterodimer-Subunit Peptides for Fusion
  • the invention also includes an affinity matrix composition useful for purifying polypeptides, such as fusion proteins containing selected polypeptides expressed in tandem with a first heterodimer-subunit peptide
  • the matrix includes a solid support having attached thereto a heterodimer-subunit peptide that is capable of forming an ⁇ -helical coiled- coil with another, complementary heterodimer-subunit peptide expressed as part of a fusion protein
  • the affinity feature of the matrix is based on selective dimerization of the complementary heterodimer-subunit peptides The selective dimerization process for purification is shown schematically in Figures
  • FIG. 1A-1F Figure IA shows a selective dimerization mat ⁇ x 30 consisting of solid phase matrix 32 and heterodimer-subunit peptide 34
  • matrix 32 is conjugated to peptide 34 through a spacing group 36
  • the presence of such a group may be particularly advantageous when the protein to be purified on the column is a large or sterically bulky protein
  • Solid phase matrix 32 may be formed by any of a number of suitable materials known in the art
  • the solid phase material will consist of spherical particles such as aminopropyl-de ⁇ vatized glass particles, capable of forming a packed matrix in a chromatography column, however, the solid phase may be formed by a continuous surface or by non-spherical particles or beads
  • the method used for conjugation of the peptide to the solid phase will be readily determined by the skilled practitioner, based on the type of mat ⁇ x. the type of spacing group, if present, and the desired peptide linkage
  • the fusion protein is pu ⁇ fied by first obtaining, from the host cell expression system, a suspension containing the fusion protein using methods known in the art (e g , Ausubel, et al )
  • This suspension is typically an impure solution containing impurities, such as other proteins, in addition to the desired fusion protein
  • the affinity mat ⁇ x 30 binds to fusion protein 38 containing selected polypeptide 40. and heterodimer-subunit peptide 42. complementary to the immobilized heterodimer-subunit peptide 34 Also shown are non-binding components of the solution such as impurities 44 that do not specifically interact with the heterodimer- subunit peptide on the column These impurities are removed from the column milieu by extensive washing of the column Exemplary wash conditions include a salt wash step with a solution having a pH of between about 5 5 and about 8 0 (e g .
  • Suitable salts include sodium chloride (NaCl), potassium chloride (KCl), sodium acetate (NaOAc).
  • Suitable organic solvents include methanol (MeOH), ethanol (EtOH), isopropanol, tetrahydrofuran (THF), t ⁇ fluoroethanol (TFE), and acetonitrile ALthough the order in which the salt and organic washes are performed is not critical, if the organic wash is done first, the column should be rinsed well with an aqueous solution (e g , water) before the start of the salt wash, to remove as much of the residual organic solvent as possible For this reason, it is generally preferable to do the salt wash first, followed by the organic wash, followed by a final aqueous rinse before the desired protein is eluted from the column.
  • aqueous solution e g , water
  • Figure IC shows a schematic of the column matrix after a wash step. Impurities shown in Figure IB are now absent from the column milieu. Fusion proteins 38 are now bound to the column through an interaction between immobilized heterodimer-subunit peptide 34 and complementary subunit 42 that forms a part of fusion protein 38.
  • Figures ID and 1E-F show alternative means of eluting selected protein 40 from the column (i.e. , means for releasing the selected polypeptide from the matrix).
  • Figure IE depicts a scheme in which the fusion polypeptide 38 is eluted from the column by washing with a buffer, such as a highly ionic buffer, that disrupts the interactions between the subunits of the coiled-coil heterodimer. As illustrated, fusion protein 38 has been removed from the column by contact with such a buffer.
  • a buffer such as a highly ionic buffer, that disrupts the interactions between the subunits of the coiled-coil heterodimer.
  • fusion protein 38 has been removed from the column by contact with such a buffer.
  • a suitable ionic buffer is 0.5 M guanidium chloride.
  • the fusion protein may be removed by eluting with a solution having a pH of less than about 3.0 and containing between about 20% and about 80% (e.g. , 50%) of a suitable organic solvent (e.g.. acetonitrile).
  • a suitable organic solvent e.g.. acetonitrile
  • the low pH of such an elution solution results in protonation of the negatively-charged residues (e.g.. Glu). disrupting the ionic interactions that stabilize the heterodimer.
  • the fusion protein is eluted with a solution having a pH of more than about 10.0 and containing between about 20% and about 80% (e.g. , 50%) of a suitable organic solvent (e.g. , acetonitrile).
  • the high pH of such an elution solution results in neutralization of the positively-charged residues (e.g., Lys), disrupting the ionic interactions that stabilize the heterodimer.
  • selected polypeptide 40 may be cleaved (Fig IF) from complementary heterodimer-subunit peptide 42 by a peptide cleavage reaction, such as by enzymatic cleavage.
  • selected polypeptide 40 is directly removed from the column by cleavage in situ, as by enzymatic cleavage. As illustrated in Figure ID, following cleavage, the polypeptide divides into complementary subunit portion 42, which remains bound as a coiled-coil heterodimer to column matrix heterodimer-subunit peptide 34, and selected protein or polypeptide 40.
  • the selective dimerization method of the invention is a useful and efficient means for purifying proteins, particularly fusion proteins, containing a heterodimer-subunit peptide capable of forming an ⁇ -helical coiled-coil in accordance with the present invention.
  • Examples 8 and 12 describe the preparation and use of an affinity matrix designed for purification of polypeptides containing a coil peptide including exemplary coupling protocols to attach one of complementary heterodimer-subunit peptides to the matrix, preparation of an affinity column using the derivatized mat ⁇ x, analysis of complementary heterodimer-subunit peptides and/or suspensions containing various contaminants along with such peptides as fusions with selected proteins run through such a column, and removal of the selected peptides and/or fusions from the column
  • Example 1 1 describes results of Western and ligand blot analyses of proteins purified using the above-described method
  • Figure 16A shows an image of a gel stained with Coomassie blue showing the relative purity of PAK (128- 144)/E-co ⁇ l pu ⁇ fied using a K-coil affinity column
  • the gel was blotted and subjected to Western blot and ligand blot analyses as described in the Example
  • the results, shown in Figure 16B show that methods of the invention can be successfully used to effectively purify recombinant fusion proteins or polypeptides
  • the invention includes synthesis and use of a reagent for detecting expressed proteins, in particular fusion proteins having a heterodimer-subunit peptide capable of forming an ⁇ -helical subunit in conformance with the invention
  • the reagent can be used in a number of research and clinical applications, including, but not limited to detection of expression of fusion proteins, ELISA applications, radioimmunoassays and the like
  • the detection reagent may also form part of a kit for detecting the expression of a selected polypeptide, where the kit also includes an expression vector that includes a DNA region encoding a heterodimer-subunit peptide capable of forming an ⁇ -helical coiled-coil heterodimer in accordance with the present invention
  • the detection reagent includes (i) a heterodimer-subunit peptide designed and formed in accordance with the methods described herein, and (n) a reporter molecule The reporter molecule may be coupled to the heterodimer subunit peptide (e
  • reporter molecule may be inco ⁇ orated into the heterodimer subunit, for example as a radioactive amino acid Radioactive amino acids are commercially available from a number of sources and can be introduced into the heterodimer-subunit peptide during chemical or biological synthesis, according to methods well known in the art
  • the reporter molecule can be selected from a number of reporter molecules well known in the art. including but not limited to radioactive groups, fluorescent tags enzymes and the like Use of enzyme markers and methods of inco ⁇ orating them into biological detection systems are known in the art (see, for example, Ausubel, et al ( 1988).
  • the reporter molecule is attached to a coupling position in the heterodimer- subunit peptide that does not interfere with heterodimer formation
  • One preferred position is position f of the subunit
  • Example 13 provides details for forming a heterodimer-subunit peptide reporter molecule in accordance with the invention
  • Figure 19 depicts a reporter molecule in a solid phase (ELISA) assay in a specific embodiment used to detect the presence of expressed proteins in accordance with the present invention
  • the reporter molecule is employed in an ELISA detection system used to quantitatively detect the presence of a heterodimer-subunit fusion protein, such as the PAK pili antigen/E-coil fusion protein described in Example 9
  • Figure 19 shows a schematic diagram of an ELISA detection system to detect the a fusion protein formed from a 17 amino acid Pseudomonas aeruginosa strain PAK pilin- de ⁇ ved peptide
  • a solid phase such as plate 50 is coated with a capture molecule such as PAK pilin peptide-specific antibody PK99H 52, according to standard methods known in the art
  • a sample containing fusion protein 54 is contacted with the plate under conditions that promote binding of the protein to antibody 52
  • fusion protein 54 is composed of a heterodimer-subunit peptide 56 and PAK pihn peptide 58 Following washing of the plate to remove unbound proteins, detection reagent 60, composed of a complementary heterodimer-subunit fusion protein, such
  • the reagent can be used to detect the presence of a complementary heterodimer-subunit peptide-containing fusion protein in a gel or on a blot likewise the reagent can be used to detect expression of the fusion protein by plaques or in cell expression systems, according to methods known in the art
  • Abbreviations used in the Examples are t-BOC, tertiary butoxycarbonyl, DCM, dichloromethane, DIEA, diisopropylethylamine, DCC, dicyclohexylcarbodimide, DMF. N,N-d ⁇ methylfo ⁇ nam ⁇ de. Gnd, guanidinium, HF, hydrogen fluoride. DCU, dicyclohexylurea.
  • BSA bovine serum albumin
  • PBS Phosphate-buffered saline
  • Plasmid pASK40 (Skerra, et al , 1991) was used for plasmid construction for E coli expression
  • Yeast expression plasmid pIC-9 (Invitrogen) was used for plasmid construction for yeast expression
  • Restriction enzymes and DNA modifying enzymes were purchased from GIBCO BRL (Gaithersburg, MD) Deoxyribonucleotides were prepared on an automatic DNA synthesizer Model 380A (Perkm-Elmer Applied Biosystems Division. Foster City. CA) Isolation of plasmid DNA and routine manipulations were carried out according to standard procedures (Sambrook, et al , 1989.
  • Proteins resolved on an SDS-PAGE gel were transferred to nitrocellulose membrane as described above The proteins on the membrane were denatured by soaking the membrane for 1 hr in PBS-ED buffer (20 mM potassium phosphate, pH 7 9. 200 mM KCl 1 mM EDTA.
  • Peptides were chemically-synthesized by solid-phase peptide synthesis using a benzhydryl amine-hydrochlo ⁇ de resin on an Applied Biosystems (Foster City, CA) peptide synthesizer Model 430A with conventional V-f-butyloxycarbonyl (t-Boc) chemistry as described previously (Hodges, et al , 1988)
  • the peptides were cleaved from the resin by reaction with hydrofluoric acid (HF, 20 ml/g resin) containing 10% anisole and 2% 1 ,2- ethanedithiol for 1 hour at -5°C to 0°C
  • the crude reduced peptides were purified by reversed-phase high performance liquid chromatography (RPC) and a "SYNCHROPAK" RP-P semi-preparative C, 8 column (250 x 10 mm inner diameter. 6 5 ⁇ m particle size, 300 ⁇ pore size, SynChrom, Lafayette, IN) with a linear AB gradient of 0 5 % B/min and 2 ml/min, where solvent A was 0 05 % trifluoroacetic acid (TFA) in water and solvent B was 0 05 % TFA in acetonitrile
  • RPC reversed-phase high performance liquid chromatography
  • SYNCHROPAK RP-P semi-preparative C, 8 column
  • amino acid composition and mass analysis were consistent with the designed sequence
  • purified peptides were hydrolyzed in 6 N HCl containing 0 1 % phenol at 100°C for 24 hours or 1 hour at 160°C in evacuated sealed tubes
  • Amino acid analysis was performed on a Beckman model 6300 amino acid analyzer (Beckman, San Ramon, CA)
  • the correct primary ion molecular weights of the reduced peptides were confirmed by plasma deso ⁇ tion time of flight mass spectroscopy on a BIOION-20 Nordic (Uppsala, Sweden)
  • Circular Dichroism Measurements Circular dichroism (CD) spectra were recorded at 20°C on a Jasco J-500C spectropola ⁇ meter (Jasco, Easton, MD) equipped with a Jasco DP-500N data processor and a Lauda (model RMS) water bath (Brinkmann Instruments. Rexdale. Ontario, Canada) for control of the temperature of the cuvette. Constant N, flushing was employed. The instrument was routinely calibrated with an aqueous solution of recrystallized d- ⁇ 0-( + )- camphorsulfonic acid at 290 nm. Molar ellipticity at 200 nm is reported as mean residue molar ellipticity ([t?] 22 o. deg»cnr»dmol ') and calculated from the equation:
  • [ ⁇ ] ⁇ is the ellipticity measured in degrees
  • mrw is the mean residue molecular weight (molecular weight of the peptide divided by the number of amino acid residues)
  • c is the peptide concentration in grams per milliliter
  • / is the optical path length of the cell in centimeters.
  • CD spectra were the average of four scans obtained by collecting data at 0.1- nm intervals from 250 to 190 nm.
  • Peptide concentrations were determined by amino acid analysis. The pH was measured at room temperature.
  • EXAMPLE 3 Heterodimer vs. Homodimer Formation Peptides 0993 (SEQ ID NO:26) and 0994 (SEQ ID NO:27) were synthesized as described in Example 1. CD spectra of peptide mixtures of different ratios of the first heterodimer-subunit peptide 0993 (SEQ ID NO: 26) and the second heterodimer-subunit peptide 0994 (SEQ ID NO:27) were measured as described in Example 2, to determine the degree of heterodimer vs. homodimer formation. The peptides were suspended in a solution containing 0. 1 M KCl and 50 mM potassium phosphate buffer, pH 7 at 20°C (reaction buffer). The total peptide concentration (sum of 0993 and 0994 concentrations) was 20 ⁇ M.
  • the gradient employed during this purification step was a 1 % B/minute gradient (Solvent A: 5 mM NaH : PO 4 /20% acetonitrile. pH 5. Solvent B: 5 mM NaH,PO 4 /20% acetonitrile, 1 M NaCl, pH 5).
  • the isolated conjugate was then desalted using a reversed-phase column and a standard 2 % B gradient (vide supra). In this way, pure conjugate was obtained which was shown through mass spectrometric analysis to be the desired product (MW calc: 7432.0, Found, 7432.4).
  • EXAMPLE 5 Recombinant Coil Gene Synthesis The E-coil gene was synthesized as follows. A DNA sequence was constructed from back translation of the de novo designed coil protein sequence using a codon bias based the on highly expressed Pichia pastoris genes (Koutz, et al. , 1989). To avoid creation of excess restriction sites, four overlapping oligonucleotides were synthesized and purified by electrocution on a 12% polyacrylamide gel containing 7M urea. The coil DNA fragments were synthesized using Klenow fragment of E. coli DNA Polymerase I as shown in Figs. 5A through 5G.
  • Oligonucleotides 1 SEQ ID NO:6 and 2 (SEQ ID NO:7) were annealed together by their complementary sequences and the remaining complementary strands were synthesized by Klenow fragment as illustrated in Figures 5 A and 5B.
  • An identical protocol was carried out for oligonucleotides 3 (SEQ ID NO:8) and 4 (SEQ ID NO:9) (Figs. 5D and 5E).
  • the double stranded fragments were then digested with restriction enzyme Alw21 I to create sticky ends at one end of each fragment (Figs. 5C and 5F), and the two fragments were ligated with T4 DNA Ligase to generate a double-stranded fragment (Fig. 5G) of 177 base pairs.
  • the fragment was excised, electroeluted from the gel slice, and digested simultaneously with EcoRI and BamHl to produce a 166bp fragment suitable for cloning into a plasmid vector.
  • Expression vector pASK40 was modified at its polycloning site to produce plasmid pRLD.
  • pASK40 was digested with EcoRI and the resulting 5' protruding ends were blunted with Mung-Bean Nuclease.
  • the blunted plasmid was then digested with Hindl and religated with a synthetic 43 bp annealed deoxyribonucleotide fragment formed by annealing oligonucleotides 5 (SEQ ID NO: 10) and 6 (SEQ ID NO: 1 1 ).
  • This modification which was verified by double stranded DNA sequencing, was designed to shift the reading frame and make pASK40's cloning site more consistent with that of Pichia pastoris vectors.
  • the synthesized E-coil gene was double digested with EcoRI and BamHl as described above and force-cloned into pRLD digested with EcoRI and Bglll to produce the E. coli E-coil vector pRLD-E ( Figure 5H).
  • the ligated plasmids were transformed into E. coli expression strain JM83 by heat-shock treatment (Yanisch-Perron, et al., 1985) and plated on selective LB plates supplemented with carbenicillin (100 ⁇ g/ml). Insertion of the coil gene was verified by restriction digest analysis and subsequent DNA sequencing of minipreped plasmid DNA of successful transformants.
  • coli cells harboring pRLD-E were grown at 25 °C with shaking in LB medium containing 100 ⁇ g/ ⁇ l of carbenicillin to an A 550 of 0.5. Expression was induced by adding isopropylthiogalactoside (IPTG) to a final concentration of ImM and incubating for 3 hours at 25 °C with shaking.
  • IPTG isopropylthiogalactoside
  • Expressed protein was obtained by a modified osmotic shock treatment (Ausubel, et al , 1988) Cells were collected at 4000 xg for 10 minutes at room temperature and resuspended in 100 mM T ⁇ s-Cl pH 8 0 containing 5mM EDTA and 20% sucrose (TES buffer) at 80ml per gram wet weight The cells were shaken at 200 ⁇ m at room temperature for 10 minutes The suspension was then centrifuged again and the pellet resuspended in 5 mM ice-cold MgS0 4 (80 ml per gram wet weight) This was shaken for 30 minutes on ice and subsequently centrifuged at 8000 xg at 4°C for 10 minutes The supernatant constituting the pe ⁇ plasmic fraction was further purified using the coil affinity column, as described in Example 8, below
  • E-coil gene described above was used as a template for the polymerase chain reaction (PCR, Saiki, et al , 1988, Mul s. 1987, Mulhs, et al , 1987) to generate an E-coil gene with a 5' EcoRI sue and 3' NotI site Synthetic Ecop ⁇ (SEQ ID NO 12) and Notp ⁇ (SEQ ID NO 13) oligonucleotides were used as primers at an annealing temperature of 50°C for 30 cycles
  • the PCR amplification product was cloned into plasmid pIC9 (Invitrogen, San Diego, CA), which contained the alpha mating factor leader sequence for secretion into the medium, generating Pichia E-coil vector p9CE Successful ly- gated plasmids were transformed into E coli cloning strain TOP10 F' and plated on LB/Carbenicil n ( 100 ⁇ g/ml) plates Plasmid DNA was prepared from selected colonies and checked for the correct size of insert by digestion with restriction enzymes and confirmed by DNA sequence analysis
  • Verified yeast plasmids were transformed into Pichia pastoris strain GS 1 15 as described (Cregg, et al , 1985) GS1 15 was cultured in YPD (1 % peptone, 2% yeast extract, 2% dextrose) media (Romanos, et al , 1991) at 30°C with shaking to an A ⁇ of 0.25 and harvested by centrifugation
  • the cell pellet underwent a series of washes with dH 2 0 and IM aqueous sorbitol, followed by IM aqueous sorbitol containing 25mM EDTA and 100 mM dithiothreitol (SED buffer) and again with IM sorbitol
  • the final pellet was resuspended in IM aqueous sorbitol containing 10 mM sodium citrate buffer, pH 5 8, and ImM EDTA (SCE buffer) and treated with zymolase at a final concentration of 0 3mg/m
  • plasmid p9CE DNA was digested with restriction enzyme Bglll to generate two linear fragments, confirmed by electrophoresis on a 0 7% agarose gel.
  • Newly-prepared spheroplasts were incubated with digested DNA for 10 minutes at room temperature.
  • a fresh solution of 20% PEG with 10 mM CaCl : and 10 mM Tris-HCl, pH 7.5 (PEG/CaT) was added and incubation continued for another 10 minutes.
  • Cells were harvested at 750 xg at room temperature and the supernatant aspirated off The pellet was resuspended in SOS media (1 M sorbitol, 0.3X YPD media, 10 mM CaCU) and the cells were allowed to recover for 20 min before addition of IM aqueous sorbitol and plating on selective RDB plates (1 M Sorbitol, 1 % dextrose, 1.34% yeast nitrogen base, 4 x 10-5 % biotin and 0.005 % amino acids without histidine).
  • Recombined clones were identified by growth in the absence of histidine, and displacement of the chromosomal AOX1 gene was distinguished by growth on selective minimal methanol media ( 1.34% yeast nitrogen base, 4 x 10-5% biotin, 0.5 % methanol).
  • Clones that had undergone successful integration of the linerarized plasmid fragment were identified by PCR screening (Clare, et al. , 1991) using vector-specific primers (SEQ ID NO: 14, SEQ ID NO: 15).
  • Recombinant yeast clones exhibiting a methanol sensitive phenotype were cultured in BMGY media (peptone, yeast extract, yeast nitrogen base, biotin. glycerol) to an A,TM of 1.0.
  • BMGY media peptone, yeast extract, yeast nitrogen base, biotin. glycerol
  • A,TM methanol
  • the carbon and energy source was subsequently changed to methanol (BMMY media) to allow for induction using the AOX 1 promoter (Ellis, et al. , 1985).
  • the culture supernatant was harvested by centrifugation at 4000 xg and filtered through a 0.2 ⁇ m filter in preparation for coil affinity purification, described in Example 8, below.
  • This section describes preparation and use of an affinity matrix designed for purification of polypeptides containing a coil peptide.
  • the affinity purification procedure is based on selective dimerization of a heterodimer-subunit peptide immobilized on a solid phase to a proteins or peptides containing a complementary heterodimer subunit.
  • polypeptides selected for purification are fusion peptides designed and synthesized to contain a heterodimer-subunit peptide complementary to the immobilized peptide
  • one of a pair of complementary peptide subunits is synthesized as a linear peptide according to methods known in the art purified, then conjugated to a solid matrix through one of the residues present in the peptide Preferably, a cysteine residue will be available for conjugation of the peptide
  • a Preparation of Selective Dimerization Affinity Matrix 1 Preparation of Peptides
  • Complementary peptides were designed as described above to form a stable ⁇ -helical coiled-coil heterodimer
  • one of the peptide subunits was synthesized to include a spacing group, shown as the C-terminal 5 ammo acids in SEQ ID NO 27, and to have a C-terminal cysteme-amide for convenience of chemical coupling to the matrix
  • the following pair of complementary peptides was designed and prepared as follows
  • SEQ ID NO 27 (Peptide 0994) Ac-KVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGGGnLC-Amide gabcdefgabcdefgabcdefgabcdefgabcdefgabcdefgabcdef
  • peptides were synthesized on a solid phase peptide synthesizer (430A Applied Biosystems Inc , Foster City, CA) using standard t-BOC chemistry Peptide was cleaved from the synthesis resin by treatment with hydrogen fluoride Purification of each peptide was carried out using reversed phase high performance liquid chromatography (RP-HPLC)
  • the resin was next stirred for 25 minutes in a solution of bromoacetic acid dissolved in DCC/DCM solution, prepared by dissolving 138 mg ( 1 0 mmol) of Bromoacetic acid (Aldrich Fine Chemicals Catalog #25,935-7) in 2 0 ml of a 0 5 M DCC/DCM solution stirring for fifteen minutes and filtering to remove insoluble DCU
  • the resin was again drained and washed three times each with 5 0 ml DCM, DMF, MeOH, DCM
  • Peptide 0994 SEQ ID NO 27, 26 mg, 6 15 ⁇ mol
  • the peptide resin solution was stirred for 1 5 hours, after which the resin was drained and washed with 5 0 ml H 2 0
  • Figure 7B shows a chromatogram of the injection flow through fraction analyzed by RP-HPLC under the conditions described above, after injecting peptide 0993 onto the selective dimerization affinity column described in Part C, above Absence of a peak at 25 min shows that substantially all loaded peptide bound to the column
  • Figure 8 shows a chromatogram of the 60% acetonitrile wash fraction of the affinity column The presence of only a minor peptide peak at the peptide 0993 position indicates that the peptide was not eluted by the washing procedure
  • Figure 9 shows a chromatogram of the GndHCl elution fraction The major peak eluting at approximately 25 minutes is peptide 0993, indicating that 0 5 M GndHCl is effective to elute this fraction
  • the affinity column was equilibrated in 10 mM phosphate pH 6 5 at a flow rate of 0 2ml/mm
  • Figure 18 shows RP-HPLC chromatograms of purification of recombinantly expressed PAK-pili-E-coil peptide from crude penplasmic extract All HPLC runs were carried out as described above HPLC runs are indicated as follows a, pre-elution, b. crude penplasmic extract, c, break-through of penplasmic extract, d, 0 5 M KCl wash, e, 80% acetonitrile wash, f first elution wash, g. second elution wash
  • the excised band was placed in a spin filter column (BioRad. Richmond. CA) and placed on liquid nitrogen for 5 minutes The filter was then placed into an eppendorf tube and spun at top speed ( ⁇ 14.000 ⁇ m) in a microfuge Following ethanol precipitation, the eluate was treated with T4 polynucleotide kinase at 37°C for 30 minutes and the enzyme inactivated at 65°C for 15 min The phosphorylated fragment was then cloned into shrimp alkaline phosphatase-treated, EcoRl- digested pRLDE and transformed into E coli strain JM83 to produce pRLDE-PAK clones on LB/Carbenicillin plates Orientation and sequence of the insert was verified by restriction analysis and sequencing of mi preped DNA plasmid Expression and purification of the PAK/E-coil fusion protein was carried out as previously described for the E-coil protein EXAMPLE 10 Green Fluorescence Protein/E-Coil Fusion A Green Fluorescence
  • Plasmid pRLDE-GFP was constructed by digesting the eluted DNA fragment with EcoRI and Iigating the DNA into shrimp alkaline phosphotase-treated, £c ⁇ /?7-d ⁇ gested pRLD-E pRLDE-GFP was then transformed into E coli strain TOP 10 F' Orientation of the gene was determined by direct detection of fluorescent colonies on LB/CA/IPTG transformation plates ( 100 ⁇ g/ ⁇ l carbenicillin, 0 8mM IPTG) The sequence of the gene was confirmed by rest ⁇ ction analysis and sequencing Expression and purification of the protein was earned out as previously described
  • the gel was blotted and subjected to Western Blot analysis as described above Monoclonal antibody PK99H, raised against the Pseudomonas aeruginosa strain K pi (PAK pili), was diluted with TTBS (1 500). and incubated with the blots for one hour at room temperature with gentle shaking The blots were then washed three times with TTBS, and a goat anti-mouse IgG(H + L)-alkahne phosphatase conjugate (Jackson Laboratories) diluted 1 10.000 with TTBS was incubated with the blots as described above
  • Ligand-blots were performed to evaluate the ability of biotm-labelled (bt) K-coil to act as a probe
  • a gel as shown in Figure 16A was blotted as described above, and biotin-labelled K-coil ( 1 mg/ml) was diluted 1 500 in PBS-ED blocking buffer and incubated with the blots at room temperature for 1 hr with gentle agitation The membranes were washed three times. 10 minutes per wash
  • Strepavidin-alkahne phosphatase conjugate (Jackson ImmunoResearch Laboratory) was diluted ( 1 2500, vol/vol) with PBS-ED blocking buffer and incubated with the blots at room temperature for 1 hr with gentle agitation The blots were washed 3 times with PBS-ED followed by a final wash with substrate buffer (100 mM Tris-HCl pH 9 5.
  • Lanes 2-3 and 8-9 were probed using the ligand blot technique, while lanes 5-6 and 1 1 - 12 were probed using the Western blot technique
  • Lanes 1 , 4. 7 and 10 contained size markers (Rainbow marker. 2-45 kDa), lane 2 bt K-coil binding to crude PAK( 128- 144)/E-co ⁇ l penplasmic prep, lane 3 bt K-coil binding to PAK( 128- 144)/E-co ⁇ l purified by K-coil affinity column, lane 5 monoclonal antibody , PK99H. binding to crude pak ( 128-144)/E- coil penplasmic prep, lane 6.
  • a single colony of E coli strain JM83 harboring the recombinant expression plasmids was inoculated in 5 ml LB medium contain 100 ⁇ g/ml carbenicillin (Sigma) and grown at 37 °C overnight The overnight culture (2 ml) was sub-inoculated in 200 ml of mini-medium (0 67- NdH ,P0 , 0.3 % K,HP0 . 0 05 % NaCl. 4 mM MgS0 4 , 2 ⁇ M FeS0 4 . 0 1 % "NH 4 C1 (Cambridge Isotope Laboratories. Woburn.
  • the induced gene-expression cultures were harvested by centrifugation (4000 xg, 10 min). The pellets were resuspended in 100 ml of TES buffer containing 100 mM Tris, 10 mM EDTA and 20% sucrose and incubated at room temperature for 10 min with gentle agitation The cells were collected by centrifuging (7000 xg. 10 mm) and resuspended in 5 mM MgS0 4 The suspension was incubated in ice-cool bath for 30 min with agitation The supernatant which contained the penplasmic proteins was collected by centrifugation (5000 xg, 10 min) and stored at -20°C or freeze-dried
  • the freeze-dried expressed crude penplasmic preps were dissolved in 20 ml of 10 mM phosphate buffer (pH 6).
  • the dissolved sample (5 ml) was loaded on an affinity column conjugated with K-coil peptide after the column was washed and equilibrated with 10 mM phosphate buffer (pH 6)
  • the constant flow rate of HPLC was 0.2 ml/min
  • the column was washed by injecting 5 ml 0.5 M KCl (pH 6) in phosphate buffer 25 minutes after the sample was loaded.
  • Five ml of 80% acetonitrile in 10 mM phosphate buffer pH 6 5 were injected 25 mm after the 0.5 M KCl washing.
  • the PAK/E-coil fusion was eluted with 5 ml of 50% acetonitrile containing 0 1 % TFA in ddH : 0
  • the identity of the eluted sample was confirmed using reversed phase chromatography and mass spectrometer analysis
  • EXAMPLE 13 Detection Reagent This section describes synthesis and use of a reagent for detecting fusion proteins having a heterodimer-subunit peptide capable of forming an ⁇ -helical coiled-coil heterodimer with a complementary peptide in conformance with the invention
  • a Iodinated Reagent for detecting fusion proteins having a heterodimer-subunit peptide capable of forming an ⁇ -helical coiled-coil heterodimer with a complementary peptide in conformance with the invention
  • heterodimer-subunit peptide is formed as described above The peptide is then iodinated according to standard procedures known in the art
  • Preferably heterodimer- subunit peptide designed for iodination contains a tvrosme residue however iodination can also be accomplished by use of, e g .
  • a Bolton-Hunter Reagent which preferably attaches to lysine residues
  • lysines occupying position f of the subunit repeat unit may be so derivatized Kits containing Bolton-Hunter reagent are commercially available (e g ICN, Costa Mesa, CA)
  • Another iodination method is the "IODOGEN' method
  • 2 mCi of car ⁇ er- free Na' T 75 ⁇ l 0 5M phosphate buffer pH 7 4 and 20 ⁇ l of 1 ⁇ g/ ⁇ l peptide are added to a polypropylene test tube coated with 10 ⁇ g "IODOGEN'
  • the tube is agitated for 8 minutes and the solution is chromatographed by HPLC through a 10 x 0 46 cm C-8 reversed phase column with a pore size of 300 A (Brownlee Labs Santa Clara CA)
  • the sample material is eluted with a gradient from 0 1 % trifluoroacetic acid to 60% acetonitrile in 0 1 % trifluoroacetic acid
  • the major peak of active radio-iodinated peptide is detected by gamma-detection of fractions and is used as a reporter molecule in the method of the invention
  • Enzyme-linked Reagent An enzyme linked reagent can be made according to methods well known in the art
  • a gene for the desired reporter enzyme such as alkaline phosphatase or luciferase. is fused to a gene for the heterodimer subunit. and the expressed fusion protein purified by affinity chromatography on a complementary heterodimer-subunit peptide affinity column, according to the methods described above
  • the heterodimer-subunit peptide is chemically linked to the reporter enzyme
  • HRP horseradish peroxidase
  • HRP (6mg/ml in 0 IM sodium phosphate buffer, pH 7) is incubated for 0 5-1 hour at 30°C with 0 3-0 7 mg of cross- linking reagent (N-succimmidyl 4-(N-male ⁇ m ⁇ domethyl)cyclohexane-l -
  • the conjugated product can be pu ⁇ fied on "ULTROGEL" Ac A 44 (Pharmacia Biotech, Piscataway, NJ)
  • the HRP-conjugated heterodimer-subunit peptide acts as a reporter molecule when it is reacted with 4-am ⁇ noant ⁇ py ⁇ ne as substrate and increased absorbance at 510 nm is measured
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO: 4 :

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Abstract

L'invention porte sur des procédés, ainsi que sur les compositions correspondantes, faisant intervenir des peptides complémentaires de sous-unité hétérodimères capables de former un hétérodimère à structure α-hélicoïdale bispiralée pour une purification par affinité et la détection de protéines de fusion.
PCT/US1996/016032 1995-10-06 1996-10-04 Procede et compositions faisant intervenir un heterodimere bispirale pour la detection et la purification de proteines exprimees WO1997012988A1 (fr)

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JP9514507A JPH11512620A (ja) 1995-10-06 1996-10-04 発現されたタンパク質の検出および精製のためのコイルドコイルヘテロダイマー法および組成物
CA002234073A CA2234073C (fr) 1995-10-06 1996-10-04 Procede et compositions faisant intervenir un heterodimere bispirale pour la detection et la purification de proteines exprimees
AU72584/96A AU695679B2 (en) 1995-10-06 1996-10-04 Coiled-coil heterodimer methods and compositions for the detection and purification of expressed proteins
EP96934080A EP0854931A4 (fr) 1995-10-06 1996-10-04 Procede et compositions faisant intervenir un heterodimere bispirale pour la detection et la purification de proteines exprimees

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WO1999002708A1 (fr) * 1997-07-11 1999-01-21 Medical Research Council Proteines de fusion comprenant des structures bispiralees derivees de la proteine inhibitrice de if1 atpase bovine
WO1999002707A1 (fr) * 1997-07-11 1999-01-21 Medical Research Council Proteines de fusion comprenant des structures bispiralees
WO1999011774A1 (fr) * 1997-08-30 1999-03-11 Fluorescience Limited Polypeptides a structure bispiralee comportant un site d'addition
WO1999051635A1 (fr) * 1998-04-06 1999-10-14 Forschungszentrum Karlsruhe Gmbh Facteurs de transcription et leur utilisation
EP0984022A1 (fr) * 1998-09-04 2000-03-08 Actinova Limited Purification de proteines sous influence de la température
WO2000014107A1 (fr) * 1998-09-04 2000-03-16 Affitech As Purification de proteine tributaire de la temperature
US6271198B1 (en) 1996-11-06 2001-08-07 Genentech, Inc. Constrained helical peptides and methods of making same
WO2002067969A3 (fr) * 2001-02-21 2003-04-10 Medtronic Minimed Inc Compositions d'insuline stabilisees
US6767545B2 (en) 1998-06-12 2004-07-27 Governors Of The University Of Alberta Pseudomonas treatment composition and method
WO2005005464A2 (fr) * 2003-07-08 2005-01-20 University Of Sussex Segment de liaison peptidique
EP1668133A1 (fr) * 2003-09-05 2006-06-14 National Research Council of Canada Proteine hybride bispiralee avec domaines de recepteurs cellulaires
US7105307B2 (en) 1997-08-30 2006-09-12 Cyclacel, Ltd. Compositions and methods for screening for modulators of enzymatic activity
US7786261B2 (en) 2004-09-02 2010-08-31 National Research Council Of Canada Coiled-coil fusion proteins comprising cell receptor domains
WO2011094363A2 (fr) 2010-01-26 2011-08-04 The Regents Of The University Of Colorado Compositions à base du virus de la grippe et procédés pour des vaccins universels
US20130295121A1 (en) * 2005-04-15 2013-11-07 Macrogenics, Inc. Covalent Diabodies and Uses Thereof
WO2020093043A1 (fr) * 2018-11-02 2020-05-07 Chen Zibo Hétérodimères protéiques orthogonaux
US12281161B2 (en) 2021-07-28 2025-04-22 Trustees Of Boston University Polypeptides and uses thereof

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DE69518838T2 (de) * 1994-05-18 2001-05-03 The Protein Engineering Network Of Centres Of Excellence (Pence), Inc. Heterodimere trägerzusammensetzung von immunogenen polypeptiden und verfahren zu deren verwendung

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WOLBER V. ET AL.: "A universal expression-purification system based on the coiled-coil interaction of myosin heavy chain.", BIOTECHNOLOGY. THE INTERNATIONAL MONTHLY FOR INDUSTRIAL BIOLOGY, NATURE PUBLISHING GROUP, US, vol. 10., 1 August 1992 (1992-08-01), US, pages 900 - 904., XP002085105, ISSN: 0733-222X, DOI: 10.1038/nbt0892-900 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6271198B1 (en) 1996-11-06 2001-08-07 Genentech, Inc. Constrained helical peptides and methods of making same
US6498020B1 (en) 1997-07-11 2002-12-24 Medical Research Council Fusion proteins comprising coiled-coil structures derived of bovine IF1 ATPase inhibitor protein
WO1999002707A1 (fr) * 1997-07-11 1999-01-21 Medical Research Council Proteines de fusion comprenant des structures bispiralees
WO1999002708A1 (fr) * 1997-07-11 1999-01-21 Medical Research Council Proteines de fusion comprenant des structures bispiralees derivees de la proteine inhibitrice de if1 atpase bovine
AU740755B2 (en) * 1997-07-11 2001-11-15 Medical Research Council Fusion proteins comprising coiled-coil structures
WO1999011774A1 (fr) * 1997-08-30 1999-03-11 Fluorescience Limited Polypeptides a structure bispiralee comportant un site d'addition
US7105307B2 (en) 1997-08-30 2006-09-12 Cyclacel, Ltd. Compositions and methods for screening for modulators of enzymatic activity
GB2342652A (en) * 1997-08-30 2000-04-19 Fluorescience Ltd Polypeptides comprising a coiled-coil and an addition
GB2342652B (en) * 1997-08-30 2002-03-27 Fluorescience Ltd Polypeptides comprising a coiled-coil and an addition
WO1999051635A1 (fr) * 1998-04-06 1999-10-14 Forschungszentrum Karlsruhe Gmbh Facteurs de transcription et leur utilisation
US6767545B2 (en) 1998-06-12 2004-07-27 Governors Of The University Of Alberta Pseudomonas treatment composition and method
EP0984022A1 (fr) * 1998-09-04 2000-03-08 Actinova Limited Purification de proteines sous influence de la température
US6380365B1 (en) 1998-09-04 2002-04-30 Affitech As Temperature dependent ligand facilitated purification of proteins
WO2000014107A1 (fr) * 1998-09-04 2000-03-16 Affitech As Purification de proteine tributaire de la temperature
WO2002067969A3 (fr) * 2001-02-21 2003-04-10 Medtronic Minimed Inc Compositions d'insuline stabilisees
US6852694B2 (en) 2001-02-21 2005-02-08 Medtronic Minimed, Inc. Stabilized insulin formulations
WO2005005464A2 (fr) * 2003-07-08 2005-01-20 University Of Sussex Segment de liaison peptidique
WO2005005464A3 (fr) * 2003-07-08 2005-03-24 Univ Sussex Segment de liaison peptidique
EP1668133A1 (fr) * 2003-09-05 2006-06-14 National Research Council of Canada Proteine hybride bispiralee avec domaines de recepteurs cellulaires
EP1668133A4 (fr) * 2003-09-05 2007-08-29 Ca Nat Research Council Proteine hybride bispiralee avec domaines de recepteurs cellulaires
US7786261B2 (en) 2004-09-02 2010-08-31 National Research Council Of Canada Coiled-coil fusion proteins comprising cell receptor domains
US20130295121A1 (en) * 2005-04-15 2013-11-07 Macrogenics, Inc. Covalent Diabodies and Uses Thereof
US9889197B2 (en) * 2005-04-15 2018-02-13 Macrogenics, Inc. Covalently-associated diabody complexes that possess charged coil domains and that are capable of enhanced binding to serum albumin
WO2011094363A2 (fr) 2010-01-26 2011-08-04 The Regents Of The University Of Colorado Compositions à base du virus de la grippe et procédés pour des vaccins universels
WO2020093043A1 (fr) * 2018-11-02 2020-05-07 Chen Zibo Hétérodimères protéiques orthogonaux
CN113272317A (zh) * 2018-11-02 2021-08-17 华盛顿大学 正交蛋白异二聚体
US11820800B2 (en) 2018-11-02 2023-11-21 University Of Washington Orthogonal protein heterodimers
US12281161B2 (en) 2021-07-28 2025-04-22 Trustees Of Boston University Polypeptides and uses thereof

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EP0854931A1 (fr) 1998-07-29
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EP0854931A4 (fr) 2002-02-13
JPH11512620A (ja) 1999-11-02

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